BCL-2 family proteins, which have either pro- or anti-apoptotic activities, have been studied intensively for the past decade owing to their importance in the regulation of apoptosis, tumorigenesis and cellular responses to anti-cancer therapy. They control the point of no return for clonogenic cell survival and thereby affect tumorigenesis and host-pathogen interactions and regulate animal development. Recent structural, phylogenetic and biological analyses, however, suggest the need for some reconsideration of the accepted organizational principles of the family and how the family members interact with one another during programmed cell death. Although these insights into interactions among BCL-2 family proteins reveal how these proteins are regulated, a unifying hypothesis for the mechanisms they use to activate caspases remains elusive.

http://dipbsf.uninsubria.it/monti/BFPN%202009/bcl2-1008.pdf

The BCL-2 protein family: opposing activities that mediate cell death

Tissue homeostasis is regulated by apoptosis, the cell-suicide programme that is executed by proteases called caspases. The Bcl2 family of intracellular proteins is the central regulator of caspase activation, and its opposing factions of anti- and pro-apoptotic members arbitrate the life-or-death decision. Apoptosis is often impaired in cancer and can limit conventional therapy. A better understanding of how the Bcl2 family controls caspase activation should result in new, more effective therapeutic approaches.

The Bcl2 family: regulators of the cellular life-or-death switch.

Irrespective of the morphological features of end-stage cell death (that may be apoptotic, necrotic, autophagic, or mitotic), mitochondrial membrane permeabilization (MMP) is frequently the decisive event that delimits the frontier between survival and death. Thus mitochondrial membranes constitute the battleground on which opposing signals combat to seal the cell's fate. Local players that determine the propensity to MMP include the pro- and antiapoptotic members of the Bcl-2 family, proteins from the mitochondrialpermeability transition pore complex, as well as a plethora of interacting partners including mitochondrial lipids. Intermediate metabolites, redox processes, sphingolipids, ion gradients, transcription factors, as well as kinases and phosphatases link lethal and vital signals emanating from distinct subcellular compartments to mitochondria. Thus mitochondria integrate a variety of proapoptotic signals. Once MMP has been induced, it causes the release of catabolic hydrolases and activators of such enzymes (including those of caspases) from mitochondria. These catabolic enzymes as well as the cessation of the bioenergetic and redox functions of mitochondria finally lead to cell death, meaning that mitochondria coordinate the late stage of cellular demise. Pathological cell death induced by ischemia/reperfusion, intoxication with xenobiotics, neurodegenerative diseases, or viral infection also relies on MMP as a critical event. The inhibition of MMP constitutes an important strategy for the pharmaceutical prevention of unwarranted cell death. Conversely, induction of MMP in tumor cells constitutes the goal of anticancer chemotherapy.

Mitochondrial Membrane Permeabilization in Cell Death

The initial insight into the genetic basis of apoptosis, or programmed cell death, was gained from ingenious studies of the roundworm Caenorhabditis elegans (for review, see Horvitz 1999). These studies revealed a linear pathway whereby the products of two genes, designated Ced-3 andCed-4, were necessary and sufficient to trigger the perfectly timed and orchestrated death of 131 preordained cells during development. The relevance of this pathway to higher animals was established by the discovery of apparent mammalian orthologs of these genes and the demonstration that the mammalian Ced-3-related genes encode proteases (designated caspases) whose activities are responsible for the morphological changes characteristic of apoptosis (for review, see Hengartner 2000). The complexity of the apoptotic program began to increase with the discovery of Bcl-2, a gene whose product causes resistance to apoptosis in lymphocytes (Vaux et al. 1988; McDonnell et al. 1989). Bcl-2 was shown to correct partially the phenotype of a C. elegans mutation in Ced-9, a cell survival gene that functions upstream of Ced-4 and Ced-3 (Vaux et al. 1992). This finding suggested an apparent one-for-one correlation between the C. elegans and mammalian proand antiapoptotic pathways. However, this correlation did not explain two observations made in mammalian cells. First, the Bcl-2 protein was found on the membrane of mitochondria, which were not implicated in C. elegans apoptosis; and second, apoptotic changes could be produced in Xenopus laevis oocyte extracts only when a membrane fraction enriched in mitochondria was present (Hockenberry et al. 1990; Newmeyer et al. 1994). The complex role of mitochondria in mammalian cell apoptosis came into focus when biochemical studies identified several mitochondrial proteins that are able to activate cellular apoptotic programs directly (Liu et al. 1996; Susin et al. 1999; Du et al. 2000; Verhagen et al. 2000; Li et al. 2001). Normally, these proteins reside in the intermembrane space of mitochondria. In response to a variety of apoptotic stimuli, they are released to the cytosol and/or the nucleus. They promote apoptosis either by activating caspases and nucleases or by neutralizing cytosolic inhibitors of this process. A complex picture has emerged in which mitochondrial and cytosolic proapoptotic proteins interact with antiapoptotic proteins with each cell’s life or death hanging in the balance. This review summarizes the recent data on the expanding and complex role of mitochondria in apoptosis.

/pdf/the-expanding-role-of-mitochondria-in-apoptosis-43fk8evh9b.pdf

The expanding role of mitochondria in apoptosis

Mitochondria: In Sickness and in Health

Bax, a pro-apoptotic member of the Bcl-2 family, translocates from the cytosol to the mitochondria during programmed cell death. We report here that both gain-of-function and loss-of-function mutations can be achieved by altering a single amino acid in the Bax hydrophobic C-terminus. The properly mutated C-terminus of Bax can target a non-relevant protein to the mitochondria, showing that specific conformations of this domain alone allow mitochondrial docking. These data along with N-terminus epitope exposure experiments suggest that the C- and the N-termini interact and that upon triggering of apoptosis, Bax changes conformation, exposing these two domains to insert into the mitochondria and regulate the cell death machinery.

/pdf/conformation-of-the-bax-c-terminus-regulates-subcellular-4i2sgaxibt.pdf

Conformation of the Bax C‐terminus regulates subcellular location and cell death

Bax is a member of the Bcl-2 family of proteins known to regulate mitochondria-dependent programmed cell death. Early in apoptosis, Bax translocates from the cytosol to the mitochondrial membrane. We have identified by confocal and electron microscopy a novel step in the Bax proapoptotic mechanism immediately subsequent to mitochondrial translocation. Bax leaves the mitochondrial membranes and coalesces into large clusters containing thousands of Bax molecules that remain adjacent to mitochondria. Bak, a close homologue of Bax, colocalizes in these apoptotic clusters in contrast to other family members, Bid and Bad, which circumscribe the outer mitochondrial membrane throughout cell death progression. We found the formation of Bax and Bak apoptotic clusters to be caspase independent and inhibited completely and specifically by Bcl-XL, correlating cluster formation with cytotoxic activity. Our results reveal the importance of a novel structure formed by certain Bcl-2 family members during the process of cell death.

/pdf/bax-and-bak-coalesce-into-novel-mitochondria-associated-15czy7tzja.pdf

Bax and Bak Coalesce into Novel Mitochondria-Associated Clusters during Apoptosis

Incubation of PC12 cells with the sulfonylurea drug, glipizide (1–100 µM), increased intracellular levels of the acidic metabolites of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). The levels of these acids in the medium were decreased, indicating the presence of a sulfonylurea-sensitive organic anion transporter. In the present study, we demonstrate that the sulfonylurea-sensitive transport of acidic dopamine metabolites is unidirectional, ATP dependent, unaffected by ouabain or by tetrodotoxin and blocked by drugs that interact with the multidrug-resistance protein-1 (MRP1). However, over-expression of MRP1 did not affect transport of the acid metabolites. The pharmacological profile and ion dependence of the transporter also differs from that of known ATP-binding cassette (ABC) family members. Using microdialysis, we also demonstrated a sulfonylurea-sensitive transport process in the striatum of freely moving rats. These results show that acidic dopamine metabolites are actively secreted from dopaminergic cells into surrounding extracellular fluid by a previously undescribed transporter.

Amotz Nechushtan

Papers

Conformation of the Bax C‐terminus regulates subcellular location and cell death

Bax and Bak Coalesce into Novel Mitochondria-Associated Clusters during Apoptosis

Acidic dopamine metabolites are actively extruded from PC12 cells by a novel sulfonylurea-sensitive transporter.

Effect of glipizide on dopamine synthesis, release and metabolism in PC12 cells.